Molecular detector arrangement

ABSTRACT

A detector assembly for detecting the presence of analyte molecules, in particular, proteins, uses both Surface Enhanced Raman Scattering (SERS) and Surface Plasmon Resonance (SPR) in synergy. The excitation laser used for SERS provides scattering from a reporter dye to which an analyte molecule is attached in the vicinity of a conducting surface. Simultaneously, a second laser is provided at the critical angle to the conducting surface. The second laser causes a field to be created in the region of the analyte which enhances the Raman scattering effect.

FIELD OF THE INVENTION

The present invention relates to a molecular detector, to a carrier foruse in molecular detector and in particular to a molecular detectorassembly of carrier and detector, which uses surface, enhanced Ramanscattering.

BACKGROUND OF THE INVENTION

It is known that there are many techniques to detect the action orpresence of analyte molecules. One such technique utilises the RamanScattering (RS) effect. Light incident on a molecule is scattered and,as a result of a transfer of energy, a shift in frequency, and thuswavelength, occurs in the scattered light. The process leading to thisinelastic scatter is termed the Raman effect. The shift in frequency isunique to the analyte molecule. The RS effect, however, is very weak, soa technique preferably using colloids is known to be used to enhance theeffect. Analyte molecules placed within a few Angstroms of a metalsurface, such as silver, gold, copper or other such materials,experience a transfer of energy from the metal surface through variousmechanisms. This is known as Surface Enhanced Raman Scattering (SERS)and can be measured using conventional spectroscopic detectors.

We have appreciated the problem that the Raman scattering effect, evenusing surface enhanced Raman scattering (SERS), provides a small amountof Raman scattered radiation in comparison to normal scattering(effectively a poor signal to noise ratio).

SUMMARY OF THE INVENTION

The invention is defined in the claims to which reference is directed.An embodiment of the invention uses surface enhanced Raman scattering(SERS) to detect the presence of an analyte in a region near the surfaceusing a first laser source incident on the region, but further enhancesthe SERS effect using a second laser incident on a surface to generate afield. The field generated in the region by the second laser is usedenhance the Raman scattering effect.

The second laser incident on the surface is preferably used additionallyfor surface plasmon resonance detection (SPR) so that both SERS and SPRdetection techniques can be used simultaneously. The SPR laser thusprovides both a function of SPR and enhances the SERS effect as well.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the invention will now be described, by way of exampleonly, and with reference to the accompanying drawings, in which:

FIG. 1 shows energy levels of Raman scattering;

FIG. 2 is a schematic diagram showing a detector using the principle ofSurface-Enhanced Raman Scattering;

FIG. 3 is a schematic diagram showing a detector using the principle ofSurface Plasmon Resonance;

FIG. 4 is a schematic diagram showing a detector arrangement using acombination of Surface-Enhanced Raman Scattering and Surface PlasmonResonance according to the invention;

FIG. 5 shows an analyte carrier and detector together forming a detectorassembly according to a first, preferred embodiment of the invention;

FIG. 6 shows an analyte carrier according to a second embodiment of theinvention; and

FIG. 7 shows an analyte carrier according to a third embodiment of theinvention.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The embodiments described uses the technique of Surface Enhanced RamanSpectroscopy (SERS) in synergy with Surface Plasmon Resonance (SPR).These techniques in combination, we have appreciated, can use theincident radiation of laser used for SPR to enhance the SERS effect. Thepresent embodiments comprise two main components: an analyte carrierwhich provides an analyte region to support molecules to be analysed;and a detector which provides laser radiation to the analyte region onthe carrier and has sensors to detect radiation received from theanalyte region. Together the analyte carrier and detector comprise adetector assembly.

The detector itself can comprise various forms of laser source andsensors as described later. The embodiments of analyte carrier,appropriate to the detector can take various forms. The preferredembodiment is a microfluidic chip, but other embodiments include asuitably modified microtiter plate or a prism arrangement also asdescribed later. The analyte carrier is thus a so called “lab on chip”.Prior to describing the embodiments, the SERS and SPR effects will firstbe described by way of background.

When light is scattered from a molecule, most of the photons areelastically scattered. The majority of the scattered photons have thesame energy (and therefore frequency and wavelength) as the incidentphotons. However, a small fraction of the light (approximately 1 in 10⁷photons) is scattered at frequencies different from, and usually lowerthan, the frequency of the incident photons as shown in FIG. 1. When thescattered photon loses energy to the molecule, it has a longerwavelength than the incident photon (termed Stokes scatter). Conversely,when it gains energy, it has a shorter wavelength (termed anti-Stokesscatter).

The process leading to this inelastic scatter is termed the Ramaneffect, after Sir C. V. Raman, who discovered it in 1928. It isassociated with a change in the vibrational, rotational or electronicenergy of the molecule, with the energy transferred from the photon tothe molecule usually being dissipated as heat. It is also possible forthermal energy to be transferred to the scattered photon, thusdecreasing its wavelength. In classical terms, this interaction can beviewed as a perturbation of the molecule's electric field, which isdependent not just on the specific chemical structure of the molecule,but also on its exact conformation and environment. The energydifference between the incident photon and the Raman scattered photon isequal to the energy of a vibrational state of the scattering molecule,giving rise to scattered photons at quantised energy values. A plot ofthe intensity of the scattered light versus the energy (wavelength)difference is termed the Raman spectrum [RS]. An explanation of thedifferent energy states is shown in FIG. 1.

FIG. 2 shows how the Raman scattering from a compound or ion within afew tens of nanometers of a metal surface can be 10³ to 10⁶ timesgreater than in solution. This Surface-Enhanced Raman scattering (SERS)is strongest on silver, but is readily observable on gold and copper aswell. Recent studies have shown that a variety of transition elementsmay also give useful SERS enhancements. The SERS effect is essentiallycaused by an energy transfer between the molecules and anelectromagnetic field near the surface of a metal caused by electrons inthe metal. The precise mechanism that leads to the enhancement of Ramanscattering using SERS need not be described here and various models suchas coupling of an image of an analyte molecule to electrons in the metalare known to the skilled person. In effect, electrons in the metal layer6 supply energy to the molecule thereby enhancing the Raman effect.

The presence of a particular molecule is detected using SERS bydetecting the wavelength of scattered radiation shown as scattered beam4. The scattering is not directional and so the sensor (not shown) couldbe at any reasonable position to capture scattered radiation to measurethe wavelength, and hence energy change, of the scattered radiation. Theenergy change is related to the band gap of molecular states, and hencethe presence of particular molecules can be determined. Typically, amolecule 10 to be analysed is bound to a reporter molecule 8 foranalysis.

A different technique for measuring the presence of molecules is knownas surface plasmon resonance (SPR) and is shown in FIG. 3. The electricvector of an excitation laser beam 12 induces a dipole in the surface ofa metal layer 16. The restoring forces from the positive polarisationcharge result in an oscillating electromagnetic field at a resonantfrequency of this excitation. In the Rayleigh limit, this resonance isdetermined mainly by the density of free electrons at the surface of themetal layer 16 (the ‘plasmons’) determining the so-called ‘plasmawavelength’, as well as the dielectric constants of the metal and itsenvironment.

Molecules in an analyte absorbed on or in close proximity to the surfaceof the layer 16 experience an exceptionally large electromagnetic fieldin which vibrational modes normal to the surface are most stronglyenhanced. This is the Surface Plasmon Resonance (SPR) effect, whichenables through-space energy transfer between the plasmons in the metallayer 16 and the molecules 8 near the surface. Scattered photons maythen be measured using conventional spectroscopic detectors (not shown).

The intensity of the SPR is dependent on many factors including thewavelength of the incident light and the morphology of the metalsurface, since the wavelength of incident light should be such that theenergy matches that of the plasma wavelength of the metal. SPR can beperformed using colloidal metal particles or thin metal films. For a 5μm silver particle the plasma wavelength is about 382 nm, but it can beas high as 600 nm for larger ellipsoidal silver particles. The plasmawavelength is to the red of 650 nm for copper and gold particles, theother two metals which show SERS at wavelengths in the 350-1000 nmregion. The best morphology for surface plasmon resonance excitation isa small (<100 nm) particle or an atomically rough surface on a thin (ca.50 nm) metal film.

As shown in FIG. 3, for SPR, an excitation laser beam 12 of planepolarised light is arranged so that it impinges on the metal surface 16close to the critical angle. This critical angle is determined by therefractive index of the metal. The SPR effect produces an evanescentwave 17, an electromagnetic field, which extends approximately 400 nmfrom the metal surface. An energy transfer between this field and theanalyte molecules results in a change in the effective refractive indexof the layer 16 causing a change in the critical angle and hence achange in the intensity of refracted light 14, which can be detectedusing conventional spectroscopic devices.

Both the RS and SPR are powerful techniques which are routinely used tofollow molecular interactions or quantify molecules at extremely lowconcentrations.

The principles of operation of the embodiments of the invention areshown in FIG. 4. A key feature of the embodiments is that the SERSeffect for detecting presence of molecules is enhanced by use of anadditional incident laser source, which is preferably also used for SPRdetection. The two detector systems can operate independently, givingdiscreet or simultaneous measurements of the same analyte sample. Theeffects behave synergistically, selectively enhancing the interactionbetween the surface plasmons and the analyte molecules.

The features previously described in relation to FIGS. 2 and 3 are giventhe same numbering in FIG. 4. A first laser source, a SERS excitationlaser beam 2, is incident on a receptor molecule 10, typically anantibody which is bound to a reporter molecule 8 at a metal surface 16which is electrically conductive. The analyte molecules are typicallyprotein molecules. When an analyte molecule binds to the receptormolecule, the reporter molecule is displaced and comes close to thesurface, thereby showing an enhancement in the SERS scattering. In knownfashion, as described above, SERS scattering occurs and the scatteredradiation 4 is detected by a sensor. At the same time, a second lasersource, an SPR laser beam 12, is incident on the metal surface 16. Thesecond laser beam couples with surface plasmons, which in turn generatean electromagnetic field, which couples with vibrational energy statesof the molecules to be analysed.

The efficiency of energy transfer between the molecular system and theplasmon field is dependant upon a match between the vibrational energystates of the molecule, and the quantum energy states of the surfaceplasmons. The former is determined by the molecular structure andenvironment, and the latter by the wavelength of the excitation laserand composition and geometry of the metal particle layer. Therefore, ifthe excitation wavelength of the SPR beam 12 is varied (e.g. by using atunable laser) or the composition and thickness of the metal layer 16 isaltered, the SPR effect can be selectively optimised to maximise theSERS signal from a particular analyte molecule. The benefits of this arethat the strength of the SERS signal can be substantially increased fora given molecule (enabling more sensitive detection), and that the SPRelectromagnetic field in a region 20 can be adjusted to selectivelyenhance the signal from particular components of complex biologicalmixtures. Since the combined detector uses an artificial SPR field toenhance the fluorescence from the analyte molecules, we have named thetechnique Surface Plasmon Assisted Raman Spectroscopy (SPARS).Effectively, the second laser is used to pump energy into the excitationproduced by the first laser.

The preferred embodiment of the invention is to apply the new techniquedescribed above in a so called lab on chip device. In this arrangement,an analyte carrier is provided (which is disposable) to which a solutioncontaining the molecules to be analysed is added. The carrier is theninserted into a detector comprising two lasers (one for SERS and one forSPR excitation) and a sensor arrangement to detect the Raman scatteredradiation and optionally the SPR radiation.

The embodiments of analyte carrier will now be described, as well asdescribing the whole carrier and detector assembly.

The preferred embodiment of analyte carrier is shown in FIG. 5 and is aform of microfluidic chip. On a substrate 11 of suitable plastic, glassor other appropriate material that is transparent to radiation at thechosen wavelengths, is formed a channel layer 13 having a channel 22.Analyte in solution is introduced to the channel in the direction shownby an arrow. At a region 17 of the channel a conductive orsemiconductive layer 16 is formed. This layer is preferably one ofcopper, aluminium, silver or particularly gold. As previously described,the gold layer maybe colloidal of particle size of the order 80 nm, theparticle size within the metal colloid being chosen to provide anappropriate plasmon wavelength as already described.

A primary use of the chip is in the detection of proteins. For this use,a reporter dye is provided on the gold surface 16 having a linkingmolecule to which an antibody or similar receptor attaches, and also apeptide or similar fragment able to mimic a portion of the targetprotein. The reporter dye is initially held away from the surface bybinding to the receptor site on the antibody or receptor molecule. Onbinding of a target protein, the reporter is displaced and comes withinthe region 20 of influence of the evanescent field from the metalsurface. The reporter dye is chosen depending upon the protein to beanalysed. It is the reporter molecule that provides the SERS scatteringas enhanced by the SPR laser.

The detector into which the analyte carrier chip is inserted comprises aSERS laser 28 providing a beam 2 to the analyte and reporter moleculesat the surface region 17 of the gold layer 16. The SERS laser 28provides radiation at a wavelength chosen to match a bandgap of thereporter molecule and will vary from molecule to molecule. To provide aflexible detector, therefore, the SERS laser is preferably tunable. AsSERS scattering 4 is not directional, the sensor 26 for the scatteredradiation could be at any position. However, this sensor is preferablenot opposite the SERS laser to avoid direct radiation from the laserreaching the sensor.

A laser 27 to provide a plane polarised beam 12 for the SPR effect isprovided at the critical angle to the surface 16, and a sensor array 24positioned so as to receive the reflected beam 14. The SPR laser 27 ischosen to have a wavelength to match the surface plasmon resonance,which itself is arranged to couple with the bandgap of the reporter dyemolecules. Thus, it is also preferable that the SPR laser 27 istuneable. The sensor array 24 comprises multiple sensor, each at aslightly differing angle to the reflected beam. Accordingly, as thereporter molecule interacts with the evanescent wave from the surface 16it changes the SPR refracted radiation which can be detected as a changein angle of the refracted light. Also, as the SPR laser is tuneable, theSPR effect can be measured by sweeping the tuning of the laser andnoting the variation in the wavelength at which the refraction occursfor a given detector position when an analyte molecule attaches to thereceptor molecule.

Although shown with just one channel, the chip preferably has multiplechannels, each of which may contain a different reporter dye and/orreceptor molecule on the metal layer.

A second embodying analyte carrier is shown in FIG. 6 and comprises amodified microtiter plate. A microtiter plate is known to the skilledperson and comprises a series of wells in a substrate, typically ofplastic. Samples of an analyte are introduced to the microtiter platewells for analysis. In accordance with the embodiment of the invention,the bottom of each well, or sides, is modified to include a conductivesurface 16 onto which a reporter dye is placed. The analyte in solutionis then introduced into each well and the plate inserted into a detectoras previously described in relation to FIG. 5. The conductive surface ispreferably gold of typical thickness 50 to 80 nm as previouslydescribed. The detector arrangement can illuminate each well in turn,but preferably has an array of detectors to allow simultaneousillumination and detection from each of a plurality of wells in theplate.

A third embodiment of chip is shown in FIG. 7. In this arrangement, aprism is effectively created from a substrate 11 having a reflectivesurface 15 and a surface 19 on which sensors are mounted for externalconnection. The gold layer is provided on a side or the prism. The goldlayer, lasers and arrangement of sensors is as described in relation tothe first embodiment.

For any of the above “lab-on-a-chip” devices, there is the additionalpossibility of controlling the exact composition of the metal layer 16.Modifying the metal surface 16 with a variety of dopant atoms wouldprovide an additional means of modulating the plasma wavelength, maybeeven resulting in an electronically-controllable SPR field.

The RS and SPR components can be physically separated, with the RS laserand detector arranged ‘above’ the analyte molecules, and the SPR laserand detector arranged ‘below’ them. Alternatively, both lasers mayilluminate the detector surface from the same side. The benefit of thisfor a lab-on-a-chip application is that it provides modularity:detectors can be built in all three combinations (RS only, SPR only, andSPARS) using the same basic components.

1.-33. (canceled)
 34. A detector assembly for detecting the presence ofa molecule in an analyte comprising: an analyte carrier having aconducting surface for receipt of an analyte in an analysis region ofthe surface; a first laser radiation source arranged to provideradiation directed, in use, to the analysis region to cause Ramanscattering; a first sensor arranged to detect radiation from the firstlaser radiation source that has been scattered from the analysis regionby Raman scattering to detect the presence of the molecule; a secondlaser radiation source arranged to provide radiation, in use, to theconducting surface at an angle to the conducting surface such that afield is generated in the analysis region; wherein the first and secondlaser radiation sources and the conducting surface and wavelength of thesecond radiation source are arranged such that the field generated bythe second laser source matches a band gap of the Raman scattering andthereby causes an enhanced Raman scattering effect of radiation of thefirst laser source.
 35. A detector assembly according to claim 34,wherein the conducting surface comprises a colloidal metal film.
 36. Adetector assembly according to claim 34, wherein the metal film is oneof aluminium, copper, silver or gold.
 37. A detector assembly accordingto any one of claims 34-36, wherein the conducting surface has athickness of the order 10-100 nm.
 38. A detector assembly according toany one of claims 34-36, wherein the conducting surface has depositedthereon a reporter dye having a binding molecule for selectively bindingto an analyte molecule to be analyzed.
 39. A detector assembly accordingto claim 38 wherein the reporter dye is arranged so that, in use, thereporter dye is in the analysis region on binding with a molecule to beanalyzed.
 40. A detector assembly according to any one of claims 34-36,wherein the analyte carrier comprises a microfluidic chip.
 41. Adetector assembly according to claim 40, wherein the microfluidic chipincludes at least one channel, a portion of the channel having theconducting surface thereon.
 42. A detector assembly according to claim40, wherein the microfluidic chip includes multiple channels, eachchannel having a portion with a conducting surface thereon, eachconducting surface having a different reporter dye deposited thereon.43. A detector assembly according to any one of claims 34-36, whereinthe carrier comprises a microtiter plate.
 44. A detector assemblyaccording to claim 43, wherein the microtiter plate has one or morewells, each well having the conducting surface at a bottom portionthereof.
 45. A detector assembly according to any one of claims 34-36,wherein the carrier comprises a prism arrangement, the conductingsurface being arranged on one face of the prism.
 46. A detector assemblyaccording to any one of claims 34-36, wherein the second laser radiationsource is arranged to provide plane-polarised radiation to theconducting surface.
 47. A detector assembly according to claim 46,wherein the second laser radiation source is arranged to provideradiation at or near the critical angle to the conducting surface.
 48. Adetector assembly according to any one of claims 34-36, wherein theconducting surface has surface plasmons of a surface plasmon wavelength,and the second laser radiation source is arranged to provide radiationat substantially the surface plasmon wavelength.
 49. A detector assemblyaccording to claim 47, wherein the second laser source is arranged forsurface plasmon resonance detection, the detector assembling furthercomprising a second sensor arranged to detected radiation from the firstlaser light source refracted from the surface.
 50. A detector assemblyaccording to claim 49, wherein the second sensor comprises a singlesensor arranged to detect a change in intensity of the refractedradiation to detect the presence of the molecule.
 51. A detectorassembly according to claim 49, wherein the sensor comprises an array ofsensors arranged to detect a change in angle of the refracted radiationto detect the presence of the molecule.
 52. An analyte carrier for usein a detector assembly in which laser radiation from a first source isused to detect the presence of an analyte by Raman scattering, and laserradiation from a second laser radiation source is used to generate afield to enhance the Raman scattering, comprising: a substrate forsupporting the analyte and having optical properties chosen to match thelaser radiation from the first or second radiation sources; and aconducting surface on a portion of the substrate for receipt of theanalyte.
 53. An analyte carrier according to claim 52, wherein theconducting surface comprises a colloidal metal film.
 54. An analytecarrier according to claim 53, wherein the metal film is one ofaluminium, copper, silver or gold.
 55. An analyte carrier according toclaim 52, wherein the conducting surface has a thickness of the order10-100 nm.
 56. An analyte carrier according to claim 52, wherein theconducting surface has deposited thereon a reporter dye having a bindingmolecule for selectively binding to an analyte molecule to be analyzed.57. An analyte carrier according to claim 56, wherein the reporter dyeis arranged so that, in use, the reporter dye is in the analysis regionon binding with a molecule to be analyzed.
 58. An analyte carrieraccording to claim 52, wherein the analyte carrier comprises amicrofluidic chip.
 59. An analyte carrier according to claim 58, whereinthe microfluidic chip includes at least one channel, a portion of thechannel having the conducting surface thereon.
 60. An analyte carrieraccording to claim 58, wherein the microfluidic chip includes multiplechannels, each channel having a portion with a conducting surfacethereon, each conducting surface having a different reporter dyedeposited thereon.
 61. An analyte carrier according to any one of claims53 to 58, wherein the carrier comprises a microtiter plate.
 62. Ananalyte carrier according to claim 61, wherein the microtiter plate hasone or more wells, each well having the conducting surface at a bottomportion thereof.
 63. An analyte carrier according to any one of claims52 to 57, wherein the carrier comprises a prism arrangement, theconducting surface being arranged on one face of the prism.
 64. Adetector for detecting the presence of a molecule in an analyte on ananalyte carrier having a conducting surface for receipt of an analyte inan analysis region of the surface, comprising: a first laser radiationsource arranged to provide radiation directed, in use, to the analysisregion to cause Raman scattering; a first sensor arranged to detectradiation from the first laser radiation source that has been scatteredfrom the analysis region by Raman scattered from the analysis region byRaman scattering to detect the presence of the molecule; a second laserradiation source arranged to provide radiation, in use, to theconducting surface at an angle to the conducting surface such that afield is generated in the analysis region; wherein the first and secondlaser radiation sources and the conducting surface and wavelength of thesecond radiation source are arranged such that the field generated bythe second laser source matches a band gap of the Raman scattering andthereby causes an enhanced Raman scattering effect of radiation of thefirst laser source.
 65. A method of detecting the presence of a moleculein an analyte, comprising: providing the analyte on an analysis regionof a conducting surface; illuminating the analysis region with firstlaser radiation to cause Raman scattering; detecting radiation scatteredfrom the analysis region by Raman scattering to detect the presence ofthe molecule; simultaneously illuminating the conducting surface withsecond laser radiation at an angle to the conducting surface andwavelength such that the field generated by the second laser sourcematches a band gap of the Raman scattering to generate a field in theanalysis region; and wherein the field generated in the analysis regionenhances the Raman scattering effect.